1. Field of the Invention
The present invention relates generally to bone physiology. More specifically, the present invention relates to in vitro and in vivo models for screening compounds to prevent glucocorticoid-induced bone destruction.
2. Description of the Related Art
The adverse effects of hypercortisolism on bone have been recognized for over 60 years (1), but the precise cellular and molecular basis of these changes has remained elusive. Today, the iatrogenic form of the disease has become far more common than Cushing""s syndrome and glucocorticoid-induced osteoporosis is now third in frequency following post-menopausal and senile osteoporosis (2).
Bone loss due to glucocorticoid excess is diffuse, affecting both cortical and cancellous bone, but has a predilection for the axial skeleton. Spontaneous fractures of the vertebrae or ribs are, therefore, often presenting manifestations of the disorder (3,4). A cardinal feature of glucocorticoid-induced osteoporosis is decreased bone formation (5). In addition, patients receiving long-term glucocorticoid therapy sometimes develop collapse of the femoral head (osteonecrosis), but the mechanism underlying this is uncertain (6). Decreased bone formation, and in situ death of isolated segments of the proximal femur suggest that glucocorticoid excess may alter the birth and death of bone cells. Defective osteoblastogenesis has been reported to be linked to reduced bone formation and age-related osteopenia in the SAMP6 mouse (7). Besides the relationship between aberrant osteoblast production and osteoporosis, it has been recently shown that a significant proportion of osteoblasts undergo apoptosis (8), which raises the possibility that the premature or more frequent occurrence of osteoblast apoptosis could contribute to incomplete repair of resorption cavities and loss of bone.
Thus, the prior art is deficient in compounds that possess the advantageous properties of glucocorticoids, namely anti-inflammatory properties, but do not cause bone loss or osteoporosis. The present invention provides for methods of screening compounds to fulfill this long-standing need and desire in the art.
To demonstrate that glucocorticoid-induced bone disease is due to changes in the birth or death rate of bone cells, a murine model of glucocorticoid excess was used as well as bone biopsy specimens obtained from patients with glucocorticoid-induced osteoporosis. This invention demonstrates that glucocorticoid administration decreases bone formation rate and bone mineral density accompanied by defective osteoblastogenesis and osteoclastogenesis in the bone marrow and increases apoptosis of mature osteoblasts and osteocytes.
One object of the present invention is to provide methods to screen compounds that retain the anti-inflammatory properties of glucocorticoids yet do not result in bone loss or osteoporosis due to apoptosis of osteoblasts and osteocytes.
In one embodiment of the present invention, there is provided a method of screening for compounds that reduce the bone deteriorating effects of glucocorticoids, comprising the steps of: (a) contacting osteoblast and osteocyte cells with either a glucocorticoid alone or a glucocorticoid in combination with a test compound; and (b) comparing the number of cells undergoing apoptosis following treatment with the glucocorticoid alone or following treatment with the glucocorticoid in combination with the test compound; wherein a lower number of apoptotic cells following treatment with the glucocorticoid in combination with the test compound than with the glucocorticoid alone indicates that the test compound reduces the bone deteriorating effects of the glucocorticoid. This embodiment also includes the aforementioned method, wherein the compound has little effect on the anti-inflammatory properties of the glucocorticoid, further comprising the step of comparing the anti-inflammatory response of the glucocorticoid in combination with the test compound to the anti-inflammatory response of the glucocorticoid alone; wherein essentially equivalent anti-inflammatory responses of the glucocorticoid alone and the glucocorticoid in combination with the test compound is indicates that the test compound both reduces the bone deteriorating effects, while retaining the anti-inflammatory properties of the glucocorticoid; wherein said anti-inflammatory response is determined by models of inflammation selected from the group consisting of the adjuvant-induced arthritis model and hindlimb inflammation model.
In another embodiment of the present invention, there is provided a method of screening for glucocorticoid analogs that possess decreased apoptotic properties towards osteoblast and osteocyte cells, comprising the steps of: (a) contacting the cells with either a glucocorticoid or a glucocorticoid analog; and (b) comparing the number of apoptotic cells following treatment with the glucocorticoid or the glucocorticoid analog, wherein a lower number of apoptotic cells following treatment with the glucocorticoid analog than with the glucocorticoid indicates that the glucocorticoid analog possesses decreased apoptotic properties towards the cells. This embodiment also includes the aforementioned method, wherein the glucocorticoid analog retains anti-inflammatory properties, further comprising the step of: (c) comparing the anti-inflammatory response of the glucocorticoid in combination with a test compound to the anti-inflammatory response of the glucocorticoid alone, wherein essentially equivalent anti-inflammatory responses of the glucocorticoid alone and the glucocorticoid in combination with the test compound is indicative of a glucocorticoid analog that possesses decreased apoptotic properties while retaining anti-inflammatory properties; wherein said anti-inflammatory response is determined by models of inflammation selected from the group consisting of the adjuvant-induced arthritis model and hindlimb inflammation model.
In yet another embodiment of the present invention, there is provided a method of screening for compounds that stimulate bone development, comprising the steps of: (a) contacting osteoblast and osteocyte cells with either a glucocorticoid or a test compound; and (b) comparing the number of cells undergoing apoptosis following treatment with the glucocorticoid or the test compound; wherein a lower number of apoptotic cells following treatment with the test compound than with the glucocorticoid indicates that the test compound stimulates bone development.
In still yet another embodiment of the present invention, there is provided a method of screening for compounds that increase bone mineral density, comprising the steps of: (a) contacting osteoblast and osteocyte cells with either a glucocorticoid or a test compound; and (b) comparing the number of cells undergoing apoptosis following treatment with the glucocorticoid and the test compound; wherein a lower number of apoptotic cells following treatment with the test compound than with the glucocorticoid is indicative of a compound that increases bone mineral density.
In the above-mentioned embodiments, contacting i s selected from the group consisting of in vitro cell cultures and in vivo murine animal model and determination of apoptosis is selected from the group consisting of TUNEL, DNA fragmentation and immunohistochemical analysis.
Other and further aspects, features, and advantages of the present invention will be apparent, from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.